Electrogenic Binding of Ions at the Cytoplasmic Side of the Na+,K+-ATPase

Electrogenic binding of ions from the cytoplasmic side of the Na+,K+ ATPase has been studied by measurements of changes of the membrane capacitance and conductance triggered by a jump of pH or of the sodium ion concentration in the absence of ATP. The pH jumps were performed in experiments with membrane fragments containing purified Na+,K+ ATPase adsorbed to a bilayer lipid membrane (BLM). Protons were released in a sub millisecond time range from a photosensitive compound (caged H+) triggered by a UV light flash. The sodium concentration jumps were carried out by a fast solution exchange in experi ments with membrane fragments attached to a solid supported membrane deposited on a gold electrode. The change of the membrane capacitance triggered by the pH jump depended on the sodium ion concentration. Potassium ions had a similar effect on the capacitance change triggered by a pH jump. The effects of these ions are explained by the their competition with protons in the binding sites on cytoplasmic side of the Na+,K+ ATPase. The approximation of the experimental data by a theoretical model yields the dissociation constants, K, and the cooperativity coefficients, n, of the binding sites for sodium ions (K = 2.7 mM, n = 2) and potassium ions (K = 1.7 mM, n = 2). In the presence of magnesium ions the apparent dissociation constants of sodium increased. A possible reason of the inhibition of sodium ion binding by magnesium ions can be an elec trostatic or conformational effect of magnesium ions bound to a separate site of the Na+,K+ ATPase close to the entrance to the sodium ion binding sites.